Highly efficient vitrification method for cryopreservation of human oocytes

Abstract

Two experiments were performed to develop a method to cryopreserve MII human oocytes. In the first experiment, three vitrification methods were compared using bovine MII oocytes with regard to their developmental competence after cryopreservation: (i) vitrification within 0.25-ml plastic straws followed by in-straw dilution after warming (ISD method); (ii) vitrification in open-pulled straws (OPS method); and (iii) vitrification in <0.1 microl medium droplet on the surface of a specially constructed fine polypropylene strip attached to a plastic handle (Cryotop method). In the second experiment, the Cryotop method, which had yielded the best results, was used to vitrify human oocytes. Out of 64 vitrified oocytes, 58 (91%) exhibited normal morphology after warming. After intracytoplasmic sperm injection, 52 became fertilized, and 32 (50%) developed to the blastocyst stage in vitro. Analysis by fluorescence in-situ hybridization of five blastocysts showed that all were normal diploid embryos. Twenty-nine embryo transfers with a mean number of 2.2 embryos per transfer on days 2 and 5 resulted in 12 initial pregnancies, seven healthy babies and three ongoing pregnancies. The results suggest that vitrification using the Cryotop is the most efficient method for human oocyte cryopreservation.

Qualitative and morphometric analysis of the ultrastructure of human oocytes cryopreserved by two alternative slow cooling protocols

“…In fact, after approximately 20 years of efforts in basic and clinical research [1,2,4,14,15,[27][28][29][30][31], oocyte cryopreservation has been described to achieve high standards of clinical efficiency [3,6,7,32]. Objective clinical evidence is emerging at a relatively slow pace, because IVF centres that routinely cryopreserve oocytes are still few and, consequently, current data are too small or dishomogeneous to draw definitive conclusions.…”

“…Comparing fresh oocytes with others frozen with a CRSC involving 0.1 mol/l sucrose as an extracellular CPA, Ghetler et al [36] found massive reduction in the number of CG as a effect of cryopreservation, concluding that stored oocytes should be microinjected rather than inseminated by standard IVF to prevent possible fertilization failure secondary to zona hardening. In effect, ICSI (intracytoplasmic sperm injection) had been suggested to be the elective route to achieve fertilization in cryopreserved oocytes [27] and has now become a standard of treatment [6,7,14,29]. The question of a nonphysiological discharge of CG in cryopreserved oocytes remains controversial.…”

“…Concomitantly, embryo cryopreservation has developed as the preferred form of preservation. In the last several years, though, controlled rate slow cooling (CRSC) [3][4][5] and vitrification [6,7] approaches have rapidly advanced, bringing oocyte cryopreservation into the range of treatments available for women suffering from, or at risk of, infertility. Nevertheless, the insufficiency of clinical data is still stirring controversy on the choice of the cryopres-ervation method(s).…”

Oocyte cryopreservation: is it time to remove its experimental label?

“…In the intervening years, variations to OC methods including changes in sucrose [14][15][16] and sodium [17,18] concentrations in slow freezing media, along with the first report of successful oocyte vitrification [19] and the development of novel cryotools [20][21][22][23] have combined to provide consistently improved survival and pregnancy rates with OC. In addition, although depolymerization of the oocyte's meiotic spindle is known to occur, a number of studies have now shown that the spindle reforms in the majority of oocytes after thawing [24][25][26][27][28][29].…”

High incidence of multiple aster formation in vitrified-warmed bovine oocytes after in vitro fertilization

“…Treatment with CPA induced a transient rise of intracellular free calcium level, premature exocytosis of cortical granules, and hardening of zonae pellucidae [7,8]. Application of vitrification improved the efficacy of oocyte cryopreservation, especially in mice [9,10] and humans [11,12]. However, vitrification of oocytes from large domestic species enriched with cytoplasmic lipid droplets still requires substantial improvement [13][14][15][16][17].…”